Arrays of integrated analytical devices

ABSTRACT

Arrays of integrated analytical devices and their methods for production are provided. The arrays are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices allow the highly sensitive discrimination of optical signals using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices include an integrated diffractive beam shaping element that provides for the spatial separation of light emitted from the optical reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/353,972, filed Mar. 14, 2019, which is a continuation of U.S. Patentapplication Ser. No. 15/900,608, filed Feb. 20, 2018, now U.S. Pat. No.10,234,393, which is a continuation of U.S. patent application Ser. No.15/451,305, filed Mar. 6, 2017, now U.S. Pat. No. 9,915,612, which is acontinuation of U.S. patent application Ser. No. 14/836,629, filed Aug.26, 2015, now U.S. Pat. No. 9,606,068, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 62/042,793, filedAug. 27, 2014, the disclosures of which are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

In analytical systems, the ability to increase the number of analysesbeing carried out at any given time by a given system has been a keycomponent to increasing the utility and extending the lifespan of suchsystems. In particular, by increasing the multiplex factor of analyseswith a given system, one can increase the overall throughput of thesystem, thereby increasing its usefulness while decreasing the costsassociated with that use.

In optical analyses, increasing multiplex often poses increaseddifficulties, as it can require more complex optical systems, increasedillumination or detection capabilities, and new reaction containmentstrategies. In some cases, systems seek to increase multiplex by manyfold, and even orders of magnitude, which further implicate theseconsiderations. Likewise, in certain cases, the analytical environmentfor which the systems are to be used is so highly sensitive thatvariations among different analyses in a given system may not betolerable. These goals are often at odds with a brute force approach ofsimply making systems bigger and of higher power, as such steps oftengive rise to even greater consequences, e.g., in inter reactioncross-talk, decreased signal to noise ratios resulting from either orboth of lower signal and higher noise, and the like. It would thereforebe desirable to provide analytical systems that have substantiallyincreased multiplex for their desired analysis, and particularly for usein highly sensitive reaction analyses, and in many cases, to do so whileminimizing negative impacts of such increased multiplex.

At the same time, there is a continuing need to increase the performanceof analytical systems and reduce the cost associated with manufacturingand using the system. In particular, there is a continuing need toincrease the throughput of analytical systems. There is a continuingneed to reduce the size and complexity of analytical systems. There is acontinuing need for analytical systems that have flexible configurationsand are easily scalable.

SUMMARY OF THE INVENTION

The instant invention addresses these and other problems by providing inone aspect an array of integrated analytical devices, each devicecomprising:

a nanoscale emission volume;

a detector layer optically coupled to the nanoscale emission volume;

a diffractive beam shaping element disposed between the nanoscaleemission volume and the detector layer; and

a color filtration layer disposed between the diffractive beam shapingelement and the detector layer;

wherein light is emitted from the nanoscale emission volume by aplurality of emitters within the emission volume;

wherein the detector layer comprises a plurality of sensing regions; and

wherein the diffractive beam shaping element spatially separates thelight emitted from the nanoscale emission volume and directs thespatially-separated light through the color filtration layer to theplurality of sensing regions.

In another aspect, the invention provides an array of integratedanalytical devices, each device comprising:

a nanoscale emission volume;

a detector layer optically coupled to the nanoscale emission volume;

a diffractive beam shaping element disposed between the nanoscaleemission volume and the detector layer; and

a color filtration layer disposed between the diffractive beam shapingelement and the detector layer, wherein the color filtration layercomprises 2 to 9 color filtration elements, each color filtrationelement specific for a range of light wavelengths;

wherein light is emitted from the nanoscale emission volume by aplurality of emitters within the emission volume;

wherein the detector layer comprises a plurality of sensing regions, andwherein the sensing regions are optically coupled to the colorfiltration elements; and

wherein the diffractive beam shaping element spatially separates thelight emitted from the nanoscale emission volume into a plurality ofbeams and directs the spatially-separated light beams through the colorfiltration elements and onto the sensing regions.

In some embodiments, the above arrays further comprise an analytedisposed within the nanoscale emission volume. In specific embodiments,the analyte comprises a biological sample, in more specific embodimentsthe biological sample comprises a nucleic acid, and in even morespecific embodiments the biological sample comprises a polymeraseenzyme.

The above arrays can comprise at least 1,000, at least 10,000, at least100,000, at least 1,000,000, or even at least 10,000,000 nanoscaleemission volumes.

In another aspect, the invention provides methods for producing thearrays of integrated analytical devices disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B schematically illustrate an exemplary nucleic acidsequencing process that can be carried out using the disclosed arrays ofintegrated analytical devices.

FIG. 2 provides a schematic block diagram of an integrated analyticaldevice.

FIG. 3A provides a schematic of excitation spectra for two signal eventsand an indicated narrow band excitation illumination, while FIG. 3Bschematically illustrates the resulting detected signal based upon thenarrow band illumination of the two signal events.

FIG. 4 schematically illustrates the signal profiles for each of fourfluorescent labeling groups, overlain with each of two different colorfilter profiles.

FIG. 5 schematically illustrates an integrated analytical device fordetecting signals from a sequencing reaction, where a lens elementspatially separates light emitted from a reaction cell, and directs thelight through a color filtration layer and onto a detector layer.

FIG. 6 schematically illustrates signal traces for a two-color,two-amplitude sequence-by-synthesis reaction.

FIGS. 7A-7B illustrate two views of a simplified integrated analyticaldevice including a diffractive beam shaping element for the spatialseparation of emitted light.

FIGS. 8A-8C illustrate the effects of modification of the diffractivebeam shaping element design on, for example, the spacing between thediffractive beam shaping element and the detector layer.

FIGS. 9A-9B represent the transmission of emission light from aZMW/nanowell through a representative diffractive beam shaping elementdesign.

FIGS. 10A-10D illustrate the efficiency and effect of wavelength onemission passage through a twin diffractive beam shaping element in theabsence of color filters.

FIGS. 11A-11B illustrate the design, construction, and analysis of arepresentative nanoscale device, including a diffractive beam shapingelement for spatial separation of light transmitted from an emissionvolume.

FIG. 12A illustrates the dependence on angle of incidence of opticalrejection by a dielectric interference filter. FIGS. 12B and 12C showschematically the inclusion of a low index layer at various places in anintegrated device in order to increase the efficiency of opticalrejection.

FIG. 13 illustrates the optical properties of an exemplary absorptiondye layer.

FIG. 14 illustrates the optical properties of an exemplary dielectricstack.

FIGS. 15A-15C schematically illustrate the structure of an exemplaryhybrid laser rejection filter and the optical properties of the filter.

FIGS. 16A-16B illustrate the physical properties of an exemplarydielectric stack and the relationship between the number of stack layersand optical transmission.

FIGS. 17A-17B illustrate the optical properties of a hybrid filtercomprising a TiO₂/Al₂O₃ dielectric stack and an Aptina red1 absorptionlayer.

FIGS. 18A-18B illustrate the optical properties of a hybrid filtercomprising a TiO₂/SiO₂ dielectric stack and an Aptina red1 absorptionlayer.

FIGS. 19A-19B illustrate the decreased reflectivity achieved by twoexemplary dark mirror coatings.

FIGS. 20A-20D illustrate the layouts of integrated devices withinexemplary arrays of the disclosure.

FIG. 21 illustrates specific features of an integrated analytical devicewithin an array of exemplary devices of the invention.

FIGS. 22A-22E illustrate components of an exemplary unit cell of theinstant devices and their general features.

FIG. 23 illustrates an assembled view of the components of FIGS.22A-22E.

FIG. 24 provides a cross-sectional view of an exemplary unit cell of theinstant disclosure.

FIG. 25 illustrates a cross-sectional SEM micrograph of an exemplaryintegrated device fabricated according to the instant disclosure.

FIGS. 26A-26R show an exemplary process flow for the fabrication of anarray of integrated analytical devices comprising a diffractive beamshaping element.

DETAILED DESCRIPTION OF THE INVENTION Integrated Analytical Devices

Multiplexed optical analytical systems are used in a wide variety ofdifferent applications. Such applications can include the analysis ofsingle molecules, and can involve observing, for example, singlebiomolecules in real time as they carry out reactions. For ease ofdiscussion, such multiplexed systems are discussed herein in terms of apreferred application: the analysis of nucleic acid sequenceinformation, and particularly, single molecule nucleic acid sequenceanalysis. Although described in terms of a particular application, itshould be appreciated that the applications for the devices and systemsdescribed herein are of broader application.

In the context of single molecule nucleic acid sequencing analyses, asingle immobilized nucleic acid synthesis complex, comprising apolymerase enzyme, a template nucleic acid, whose sequence one isattempting to elucidate, and a primer sequence that is complementary toa portion of the template sequence, is observed to identify individualnucleotides as they are incorporated into the extended primer sequence.Incorporation is typically monitored by observing an opticallydetectable label on the nucleotide, prior to, during or following itsincorporation. In some cases, such single molecule analyses employ a“one base at a time approach”, whereby a single type of labelednucleotide is introduced to and contacted with the complex at a time.Upon incorporation, unincorporated nucleotides are washed away from thecomplex, and the labeled incorporated nucleotides are detected as a partof the immobilized complex.

In some instances, only a single type of nucleotide is added to detectincorporation. These methods then require a cycling through of thevarious different types of nucleotides (e.g., A, T, G and C) to be ableto determine the sequence of the template. Because only a single typenucleotide is contacted with the complex at any given time, anyincorporation event is by definition, an incorporation of the contactednucleotide. These methods, while somewhat effective, generally sufferfrom difficulties when the template sequence includes multiple repeatednucleotides, as multiple bases can be incorporated that areindistinguishable from a single incorporation event. In some cases,proposed solutions to this issue include adjusting the concentrations ofnucleotides present to ensure that single incorporation events arekinetically favored.

In other cases, multiple types of nucleotides are added simultaneously,but the nucleotides are distinguishable by the presence on each type ofnucleotide of a different optical label. Accordingly, such methods canuse a single step to identify a given base in the sequence. Inparticular, all four nucleotides, each bearing a distinguishable label,is added to the immobilized complex. The complex is then interrogated toidentify which type of base was incorporated, and as such, the next basein the template sequence.

In some cases, these methods only monitor the addition of one base at atime, and as such, they (and in some cases, the single nucleotidecontact methods) require additional controls to avoid multiple basesbeing added in any given step, and thus being missed by the detectionsystem. Typically, such methods employ terminator groups on thenucleotide that prevent further extension of the primer once onenucleotide has been incorporated. These terminator groups are typicallyremovable, allowing the controlled re-extension after a detectedincorporation event. Likewise, in order to avoid confounding labels frompreviously incorporated nucleotides, the labeling groups on thesenucleotides are typically configured to be removable or otherwiseinactivatable.

In another process, single molecule primer extension reactions aremonitored in real-time, to identify the continued incorporation ofnucleotides in the extension product to elucidate the underlyingtemplate sequence. In such single molecule real time (or SMRT™)sequencing, the process of incorporation of nucleotides in apolymerase-mediated template dependent primer extension reaction ismonitored as it occurs. In preferred aspects, the template/polymeraseprimer complex is provided, typically immobilized, within an opticallyconfined region, such as a zero mode waveguide (ZMW), or proximal to thesurface of a transparent substrate, optical waveguide, or the like (seee.g., U.S. Pat. Nos. 6,917,726, and 7,170,050 and U.S. PatentApplication Publication No. 2007/0134128, the full disclosures of whichare hereby incorporated by reference herein in their entirety for allpurposes). The optically confined region is illuminated with anappropriate excitation radiation for the fluorescently labelednucleotides that are to be used. Because the complex is within anoptically confined region, or very small illumination volume, only thereaction volume immediately surrounding the complex is subjected to theexcitation radiation. Accordingly, those fluorescently labelednucleotides that are interacting with the complex, e.g., during anincorporation event, are present within the illumination volume for asufficient time to identify them as having been incorporated. Althoughthe analyte of interest in the devices disclosed herein is atemplate/polymerase primer complex that is incorporatingfluorescently-labeled nucleotides, it should be understood that otheranalytes of interest, in particular fluorescent analytes of interest,can be monitored using the devices of the instant disclosure.

A schematic illustration of this sequencing process is shown in FIGS.1A-1B. As shown in FIG. 1A, an immobilized complex 102 of a polymeraseenzyme, a template nucleic acid and a primer sequence are providedwithin an observation volume (as shown by dashed line 104) of an opticalconfinement, of e.g., a zero mode waveguide 106. As an appropriatenucleotide analog, e.g., nucleotide 108, is incorporated into thenascent nucleic acid strand, it is illuminated for an extended period oftime corresponding to the retention time of the labeled nucleotideanalog within the observation volume during incorporation which producesa signal associated with that retention, e.g., signal pulse 112 as shownby the A trace in FIG. 1B. Once incorporated, the label that wasattached to the polyphosphate component of the labeled nucleotideanalog, is released. When the next appropriate nucleotide analog, e.g.,nucleotide 110, is contacted with the complex, it too is incorporated,giving rise to a corresponding signal 114 in the T trace of FIG. 1B. Bymonitoring the incorporation of bases into the nascent strand, asdictated by the underlying complementarity of the template sequence,long stretches of sequence information of the template can be obtained.

The above sequencing reaction can be incorporated into a device,typically an integrated analytical device, that provides for thesimultaneous observation of multiple sequencing reactions, ideally inreal time. While the components of each device and the configuration ofthe devices in the system can vary, each integrated analytical devicetypically comprises, at least in part, the general structure shown as ablock diagram in FIG. 2. As shown, an integrated analytical device 200typically includes a reaction cell 202, in which the analyte (i.e., thepolymerase-template complex and associated fluorescent reactants) isdisposed and from which the optical signals emanate. The analysis systemfurther includes a detector element 220, which is disposed in opticalcommunication with the reaction cell 202. Optical communication betweenthe reaction cell 202 and the detector element 220 is provided by anoptical train 204 comprised of one or more optical elements generallydesignated 206, 208, 210 and 212 for efficiently directing the signalfrom the reaction cell 202 to the detector 220. These optical elementsgenerally comprise any number of elements, such as lenses, filters,gratings, mirrors, prisms, refractive material, apertures, or the like,or various combinations of these, depending upon the specifics of theapplication. By integrating these elements into a single devicearchitecture, the efficiency of the optical coupling between thereaction cell and the detector is improved. Examples of integratedanalytical systems, including various approaches for illuminating thereaction cell and detecting optical signals emitted from the reactioncell, are described in U.S. Patent Application Publication Nos.2012/0014837, 2012/0019828, and 2012/0021525, which are eachincorporated by reference herein in their entireties for all purposes.

As noted above, an analyte (e.g., a polymerase-template complex withassociated fluorescent reactants) disposed within a reaction cell (e.g.,element 202 in FIG. 2) or otherwise immobilized on the surface of thedevice, emits light that is transmitted to a detector element (e.g.,element 220 in FIG. 2). For fluorescent analytes, the analyte isilluminated by an excitation light source, whereas for other analytes,such as chemilunimescent or other such analytes, an excitation lightsource may not be necessary. At least a portion of the reaction cellvolume, the emission volume, is optically coupled to the detectorelement, so that light emitted from an analyte within this volume ismeasured by the detector element. In order to maximize the number ofanalytes measured simultaneously, the size of the instant analyticaldevices are reduced as much as possible, so that the emission volumewithin each device is a nanoscale emission volume. Ideally, the opticalcoupling between the nanoscale emission volume and the detector elementis highly efficient, in order to maximize the sensitivity of the deviceand maximize the signal output. As described in further detail below,light emitted from the nanoscale emission volume can be furthermanipulated, for example by lens elements and color filtration layers,prior to reaching the detector element.

Conventional analytical systems typically measure multiple spectrallydistinct signals or signal events and must therefore utilize complexoptical systems to separate and distinctly detect those different signalevents. The optical path of an integrated device can be simplified,however, by a reduction in the amount or number of spectrallydistinguishable signals that are detected. Such a reduction is ideallyeffected, however, without reducing the number of distinct reactionevents that can be detected. For example, in an analytical system thatdistinguishes four different reactions based upon four differentdetectable signal events, where a typical system would assign adifferent signal spectrum to each different reaction, and thereby detectand distinguish each signal event, in an alternative approach, fourdifferent signal events would be represented by fewer than fourdifferent signal spectra, and would, instead, rely, at least in part, onother non-spectral distinctions between the signal events.

For example, a sequencing operation that would conventionally employfour spectrally distinguishable signals, e.g., a “four-color” sequencingsystem, in order to identify and characterize the incorporation of eachof the four different nucleotides, could, in the context of analternative configuration, employ a one-color or two-color analysis,e.g., relying upon a signals having only one or two distinct ordistinguished spectral signals. However, in such an alternativeconfiguration, this reduction in reliance on signal spectral complexitydoes not come at the expense of the ability to distinguish signals frommultiple, i.e., a larger number of different signal producing reactionevents. In particular, instead of relying solely on signal spectrum todistinguish reaction events, such an alternative configuration can relyupon one or more signal characteristics other than emission spectrum,including, for example, signal intensity, excitation spectrum, or both,to distinguish signal events from each other.

In one particular alternative configuration, the optical paths in anintegrated analytical device can thus be simplified by utilizing signalintensity as a distinguishing feature between two or more signal events.In its simplest iteration, and with reference to an exemplary sequencingprocess, two different types of nucleotides would bear fluorescentlabels that each emit fluorescence under the same excitationillumination, i.e., having the same or substantially overlappingspectral band, and thus would provide benefits of being excited using asingle excitation source. The resulting signals from each fluorescentlabel would have distinct signal intensities or amplitudes under thatsame illumination, and would therefore be distinguishable by theirrespective signal amplitudes. These two signals could have partially orentirely overlapping emission spectra, but separation of the signalsbased upon any difference in emission spectrum would be unnecessary.

Accordingly, for analytical systems using two or more signal events thatdiffer in signal amplitude, the integrated analytical devices of suchsystems can readily benefit through the removal of some or all of thosecomponents that would normally be used to separate spectrally distinctsignals, such as multiple excitation sources and their associatedoptical trains, as well as the color separation optics, e.g., filtersand dichroics, for the signal events, which in many cases, requires atleast partially separate optical trains and detectors for eachspectrally distinct signal. As a result, the optical paths for theseintegrated analytical devices are greatly simplified, allowing placementof detector elements in closer proximity to reaction cells, andimproving overall performance of the detection process for thesedevices.

Provision of a signal-producing analyte that will produce differentsignal amplitudes under a particular excitation illumination profile canbe accomplished in a number of ways. For example, different fluorescentlabels can be used that present excitation spectral profiles thatoverlap but include different maxima. As such, excitation at a narrowwavelength will typically give rise to differing signal intensities foreach fluorescent group. This is illustrated in FIG. 3A, which shows theexcitation spectra of two different fluorescent label groups (solid anddashed lines 302 and 304, respectively). When subjected to excitationillumination at the wavelength range shown by vertical lines 306, eachfluorescent label will emit a signal at the corresponding amplitude. Theresulting signal intensities at a given excitation wavelength are thenshown in the bar chart of FIG. 3B as solid lined and dashed lined bars,respectively. The difference in intensity of these two signal producinglabels at the given excitation wavelength is readily used to distinguishthe two signal events. As will be appreciated, such spectrallyindistinct signals would not be easily distinguishable when occurringsimultaneously, as they would result in an additive overlapping signal,unless, as discussed below, such spectrally indistinct signals resultfrom spectrally distinct excitation wavelengths. As will be appreciated,this same approach can be used with more than two label groups, wherethe resulting emission at a given excitation spectrum havedistinguishable intensities or amplitudes.

Similarly, two different fluorescent labeling groups can have the sameor substantially similar excitation spectra, but provide different anddistinguishable signal emission intensities due to the quantum yield ofthose labeling groups.

Further, although described in terms of two distinct fluorescent dyes,it will be appreciated that each different labeling group can eachinclude multiple labeling molecules. For example, each reactant caninclude an energy transfer dye pair that yields emissions of differingintensities upon excitation with a single illumination source. Forexample, a labeling group can include a donor fluorophore that isexcited at a given excitation wavelength, and an acceptor fluorophorethat is excited at the emission wavelength of the donor, resulting inenergy transfer to the acceptor. By using different acceptors, whoseexcitation spectra overlap the emission spectrum of the donor todiffering degrees, such an approach can produce overall labeling groupsthat emit at different signal amplitudes for a given excitationwavelength and level. Likewise, adjusting the energy transfer efficiencybetween the donor and acceptor will likewise result in differing signalintensities at a given excitation illumination.

Alternatively, different signal amplitudes can be provided by differentmultiples of signal producing label groups on a given reactant, e.g.,putting a single label molecule on one reactant while putting 2, 3, 4,or more individual label molecules on a different reactant. Theresulting emitted signal will be reflective of the number of labelspresent on a reactant and thus will be indicative of the identity ofthat reactant.

Exemplary compositions and methods relating to fluorescent reagents,such as nucleotide analogs, useful for the above purposes are describedin, for example, U.S. Patent Application Publication Nos. 2012/0058473;2012/0077189; 2012/0052506; 2012/0058469; 2012/0058482; 2010/0255488;2009/0208957, which is each incorporated by reference herein in itsentirety for all purposes.

As described above, integrated analytical devices making use of suchapproaches see a reduction in complexity by elimination of spectraldiscrimination requirements, e.g., using signal amplitude or othernon-spectral characteristics as a basis for signal discrimination.Integrated analytical devices that combine such non-spectraldiscrimination approaches with the more common spectral discriminationapproaches can also provide advantages over more complex spectraldiscrimination systems. By shifting from a “four-color” discriminationsystem to a system that distinguishes signals based upon signalintensity and color, one can still reduce the complexity of the overalloptical system relative to a conventional four-color separation scheme.For example, in an analytical operation that detects four discretereaction events, e.g., in a nucleic acid sequencing analysis, two signalevents can be provided within a given emission/detection spectrum, i.e.,emitting signals within the same spectral window, and the other twoevents within a distinct emission/detection spectrum. Within eachspectral window, the pair of signal events produce distinguishablesignal intensities relative to each other.

For ease of discussion, this concept is described in terms of two groupsof fluorescent signal events, where members of each group differ byfluorescent intensity, and the groups differ by virtue of their emissionspectrum. As will be appreciated, the use of simplified optics systems,e.g., using two detection channels for two distinct emission spectra,does not require that the emission profiles of the two groups of signalsdo not overlap or that the emission spectra of members of each groupperfectly overlap. Instead, in many preferred aspects, more complexsignal profiles can be used where each different signal event possessesa unique emission spectrum, but in a way that each signal will present asignal profile within the two detection channels that is unique, basedupon the signal intensity in each channel.

For use in the instant devices, each “emitter” in a sample should thushave a unique signal profile, as just described, in order to be properlyidentified. Samples containing a plurality of emitters can thus bereadily distinguished using the instant devices. In some embodiments,the devices distinguish 4 to 18 emitters 4 to 12 emitters, or even 4 to8 emitters. In specific embodiments, the devices distinguish fouremitters, for example the four different bases of the nucleic acidsequencing reaction.

FIG. 4 schematically illustrates the signal profiles for each of fourfluorescent labeling groups, overlain with each of two different filterprofiles. As shown, four label groups yield emission spectra 402, 404,406, and 408, respectively. While the signals from these four groupspartially overlap each other, they each have different maxima. Whensubjected to a two channel filter scheme, as shown by pass filter lines410 and 412, the signal from each label will produce a unique signalprofile between the two detection channels. In particular, signals arerouted through an optical train that includes two paths that arefiltered according to the spectral profile shown. For each signal,different levels of emitted light will pass through each path and bedetected upon an associated detector. The amount of signal that passesthrough each filter path is dictated by the spectral characteristics ofthe signal.

In the case of the above described mixed-mode schemes, detection systemscan be provided that include at least two distinct detection channels,where each detection channel passes light within a spectrum that isdifferent from each other channel. Such systems also include a reactionmixture within optical communication of the detection channels, wherethe reaction mixture produces at least three different optical signalsthat each produces a unique signal pattern within the two detectionchannels, as compared to the other optical signals.

In all cases, each signal-producing reactant is selected to provide asignal that is entirely distinct from each other signal in at least oneof signal intensity and signal channel. As noted above, signal intensityin a given channel is dictated, in part, by the nature of the opticalsignal, e.g., its emission spectrum, as well as the filters throughwhich that signal is passed, e.g., the portion of that spectrum that isallowed to reach the detector in a given channel. However, signalintensity can also be modulated by random variables, such as orientationof a label group when it is emitting signal, or other variables of theparticular reaction. Accordingly, for a signal's intensity to be assuredof being entirely different from the intensity of another signal withina given channel, in preferred aspects, this variation is accounted for.

With a reduced number of spectrally distinct signal events, thecomplexity of the optical paths for the integrated devices is alsoreduced. FIG. 5 illustrates a not-to-scale example device architecturefor performing optical analyses, e.g., nucleic acid sequencingprocesses, that rely in part on non-spectral discrimination of differingsignals, and optionally, in part on spectral distinction. As shown, anintegrated analytical device 500 can include a reaction cell 502 that isdefined upon the surface layer of the device. As shown in this drawing,the reaction cell comprises a nanowell disposed in the surface layer.Such nanowells can constitute depressions in a substrate surface orapertures disposed through additional substrate layers to an underlyingtransparent substrate, e.g., as used in zero mode waveguide (ZMW) arrays(see, e.g., U.S. Pat. Nos. 7,181,122 and 7,907,800, and also below). Itshould also be understood, however, that in some embodiments, the sampleof interest can be confined in other ways, and that the nanoscalereaction cell in those embodiments can be omitted from the analyticaldevices. For example, if a target of interest is immobilized in apattern on the surface of a device lacking separate reaction cells,binding events, or other events of interest, could be observed at thoselocations without the need for physical separation of the samples.Hybridization reactions, for example between immobilized nucleic acidsand their complimentary sequences, or binding reactions, for examplebetween antibodies and their ligands, where either member of the bindingpair can be immobilized at a particular location on the surface of thedevice, could suitably be monitored using such an approach, as would beunderstood by those of ordinary skill in the art.

Excitation illumination is delivered to the reaction cell or to theimmobilized target from an excitation light source (not shown) that canbe separate from or also integrated into the substrate. As shown, anoptical waveguide (or waveguide layer) 505 can be used to conveyexcitation light (shown by arrows in one direction, although light canbe propagated in either direction or both directions, as desired) to thereaction cell 502 or otherwise immobilized target, where the evanescentfield emanating from the waveguide 505 illuminates reactants within theillumination volume. Use of optical waveguides to illuminate reactioncells is described in e.g., U.S. Pat. No. 7,820,983 and U.S. PatentApplication Publication No. 2012/0085894, which are each incorporated byreference herein in their entireties for all purposes. The nanoscalereaction cell (also referred to herein as the “nanowell” or “ZMW”) canact to enhance the emission of fluorescence downward into the device andlimit the amount of light scattered upwards.

The emitted light, whether from a nanoscale reaction cell or from animmobilized target, is directed to the detector through an integratedoptical train 504 comprising one or more optical elements. The opticaltrain includes a lens element layer 508 to direct emitted light from anemission volume within the reaction cell to a detector layer 512disposed beneath the reaction cell. As described in more detail below,the lens element layer in the integrated analytical devices of theinstant disclosure preferably comprises a diffractive beam shapingelement that serves to separate at high efficiency the emitted lightinto at least two beams for passage through the color filtration layer510. The diffractive beam shaping element may, for example, separate theemitted light into two, three, four, or even more at least partiallyseparated beams directed onto the detector layer. Depending on theconfiguration of the diffractive beam shaping element, the split beamscan be organized in a linear fashion, or they can be arranged in anarray, for example in a 2×2 beam array or the like. Such arrangementswill typically be dictated by the configuration of the sensing regionsof the detector layer.

The detector layer typically comprises one, or preferably multiple,sensing regions 512 a-b, e.g., pixels in an array detector, for examplea CMOS detector, that are optically coupled through the diffractive beamshaping element to an emission volume within a given analytical device.Although illustrated as a linear arrangement of pixels 512 a-b, it willbe appreciated that such pixels can be arranged in a grid, n×n square,n×m rectangle, annular array, or any other convenient orientation.Exemplary arrangements are described in more detail below.

It should be understood in the context of the disclosure that the“optical coupling” of two components in a device is not intended toimply a directionality to the coupling. In other words, since thetransmission of optical energy through an optical device is fullyreversible, the optical coupling of a first component to a secondcomponent should be considered equivalent to the optical coupling of thesecond component to the first component.

Emitted signals from the reaction cell 502 that impinge on the pixels ofthe detector layer are then detected and recorded. As noted above, acolor filtration layer 510 is preferably disposed between the detectorlayer and the nanoscale emission volume, to permit different spectrallydistinct signals to travel to different associated sensing regions 512 aand 512 b in the detector layer 512. For example, the portion 510 a offilter layer 510 allows only signals having a distinct first emissionspectrum to reach its associated sensing region 512 a, while filterportion 510 b of filter layer 510 allows only signals having a distinctsecond spectrum to reach its associated sensing region 512 b.

In the context of a sequencing system exploiting such a configuration,incorporation of two of the four nucleotides would produce signals thatwould be passed through filter portion 510 a to sensing region 512 a,and blocked by filter portion 510 b. As between these two signals, onesignal would have a signal intensity higher than the other, such thatthe sensing region 512 a in detector layer 512 would be able to producesignal responses indicative of such differing signal intensities.Likewise, incorporation of the other two of the four nucleotides wouldproduce signals that would be passed through filter portion 510 b tosensing region 512 b, and blocked by filter portion 510 a. As betweenthese two signals, one signal would have a signal intensity higher thanthe other, such that the sensing region 512 b in detector layer 512would be able to produce signal responses indicative of such differingsignal intensities.

The detector layer is operably coupled to an appropriate circuitry,typically integrated into the substrate, for providing a signal responseto a processor that is optionally included integrated within the samedevice structure or is separate from but electronically coupled to thedetector layer and associated circuitry. Examples of types of circuitryare described in U.S. Patent Application Publication No. 2012/0019828.

As will be appreciated from the foregoing disclosure and FIG. 5, theintegrated analytical devices described herein do not require the morecomplicated optical paths that are necessary in systems utilizingconventional four-color optics, obviating in some cases the need forexcessive signal separation optics, dichroics, prisms, or filter layers.In particular, although shown with a single filtration layer, as noted,in optional aspects, the filtration layer could be eliminated or couldbe replaced with a filtration layer that blocks stray light from theexcitation source, e.g., a laser rejection filter layer (see below),rather than distinguishing different emission signals from the reactioncell. Even including the filtration layer 510, results in simplifiedand/or more efficient optics as compared to conventional four-colorsystems, which require either multilayer filters, or narrow band passfilters, which typically require hybrid layers or composite approachesover each subset of sensing regions, thus blocking signal from reachingone or more of the sensing region subsets at any given emissionwavelength, resulting in the detection of far fewer photons from eachsignal event. The optics configuration shown in FIG. 5, on the otherhand, only blocks a smaller portion of the overall signal light fromreaching the detector. Alternatively, such conventional systems wouldrequire separation and differential direction of all four differentsignal types, resulting in the inclusion of additional optical elements,e.g., prisms or gratings, to achieve spectral separation. Examples ofnanoscale integrated analytical devices that include spectral diversionelements (i.e., optical elements that spatially separate light based oncolor) are provided in U.S. Patent Application Publication No.2012/0021525.

FIG. 6 shows a schematic exemplar signal output for a real timesequencing operation using a two color/two amplitude signal set from anintegrated system of the invention where one trace (dashed) denotessignals associated with incorporation of A (high intensity signal) and T(lower intensity signal) bases, while the other signal trace (solidline), denotes the signals of a different emission spectrum, associatedwith G (high) and C (low) bases. The timing of incorporation and theidentity of the base incorporated, as derived from the color channel andintensity of the signal, are then used to interpret the base sequence.

Lens Elements for Spatial Separation of Emitted Light

As mentioned above, the nanoscale integrated analytical devices of theinstant disclosure include a lens element layer disposed between thenanoscale emission volume and the detector layer. The lens elements ofsuch a layer serve to direct light emitted from the nanoscale emissionvolume along two or more spatially separated optical paths at highefficiency. In addition to splitting the emitted optical signals intoseparate optical paths, the lens element can additionally collimateand/or focus the emitted light. In particular, such lens elements areideally capable of collimating emitted light with near on-axis rays, aswell as splitting the emitted light, for example prior to colorseparation by the color filtration layer. In addition, such lenselements are readily fabricated using standard techniques.

The integrated optical lens elements of the instant devices can beeither refractive lenses or diffractive lenses, depending on the opticaland physical properties desired, as would be understood by those ofordinary skill in the art. A diffractive lens may, in somecircumstances, provide improved image quality, be more easilyminiaturized, and/or be less expensive to fabricate than a comparablerefractive lens. In some cases, the lenses can include separaterefractive and diffractive components or can be hybrid lenses thatcombine both features in a single lens element.

In preferred embodiments, the lens element of the instant analyticaldevices is a diffractive beam shaping element or a variant thereof. Suchelements typically include Fresnel-like lens features. Fresnel lenses,which are also known as zone plates or Fresnel zone plates when theyfunction by diffraction rather than refraction or reflection, consist ofa series of concentric rings with a specific tapered shape, or withalternating transparent and opaque zones (also called the Fresnelzones), with respect to the incident irradiation. These structuresresult in the focusing of light passing through the device by selectiveabsorption or selective phase shifting and thus allow the device tofunction as a lens. The specific lens design depends on the radiation tobe focused, the refractive index of the material used to construct thelens, and the desired focal length, as is well known in the art. In someembodiments, the lens element of the instant devices is a modified

Fresnel lens that functions as a diffractive array focusing element.This hybrid lens element can be referred to as a diffractive beamshaping element due to its ability to spatially separate emitted lightinto a plurality of spots. While the integrated analytical devices ofthe instant disclosure will be described in various embodiments asincluding a diffractive beam shaping element, it should be understoodthat these are preferred embodiments of the devices, and that other lenselements can be included in the instant analytical devices withoutlimitation.

A variety of materials and methods can be used to fabricate the lenselements of the instant devices, as would be understood by those ofordinary skill in the art. For example, the lens elements can be formedby the etching of zones in the planar surface of a material transparentto the light of interest and the subsequent deposition of an absorbingor phase shifting material into the etched zones. For example, a phaseFresnel zone plate is a staircase approximation to a phase Fresnel lens.The efficiency of the phase Fresnel zone plate increases as the numberof levels is increased. Specifically, a two-phase Fresnel zone plate canbe shown to have a maximum diffraction efficiency of 40.5%, whereas afour-phase Fresnel zone plate has a maximum diffraction efficiency of81%. The optical efficiency of the lens element, such as a diffractivebeam shaping element, is therefore in some embodiments at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or even higher. Inpreferred embodiments, the optical efficiency is at least 40%.Techniques for designing a lens element with the desired spatialseparation capabilities are known in the art. For example, theapplication of binary-optics technology in the design of opticalelements for the manipulation of laser beams (e.g., for splitting andcombining laser beams) is described by Leger et al. (1988) The LincolnLaboratory Journal 1(2): 225. Optical ray tracing software, such as theoptical design program Zemax, can be used to design such elements.

Fresnel lenses, and variants thereof, have been incorporated intoadvanced optical devices using various techniques, for example asimaging optics in illumination systems (see, e.g., U.S. Pat. No.6,002,520), in light emitting devices (see, e.g., U.S. Pat. No.6,987,613), in solid-state imaging devices (see, e.g, U.S. Pat. No.7,499,094), in image sensors (see, e.g., U.S. Pat. No. 8,411,375), andin integrated infrared sensors (see, e.g., U.S. Patent ApplicationPublication No. 2013/0043552). The design of the lens elements of theinstant disclosure and their integration into the instant analyticaldevice arrays can be achieved using analogous approaches.

The use of a diffractive beam shaping element for spatial separation oflight in the detection pathway of the instant devices provides severaladvantages over traditional optical elements, such as reflective conesor parabolic mirrors. In particular, such diffractive beam shapingelements provide off-axis focusing of light emitted from an emissionvolume. Such elements further require minimal area, minimal pitch, andresult in minimal crosstalk between adjacent detector elements. Unlikereflective cones or parabolic mirrors, as typically used in nanoscaleintegrated analytical devices, or traditional refractive lens elements,the diffractive beam shaping elements of the instant disclosure cansimultaneously collimate and split light emitted from an emission volumeat high efficiency. Furthermore, the instant diffractive beam shapingelements are readily manufactured using standard microchip fabricationtechniques, for example using standard deposition, removal, andpatterning techniques.

Two views of a simplified exemplary integrated analytical device thatincludes a diffractive beam shaping element for spatial separation ofemitted light are shown in

FIGS. 7A-7B. FIG. 7A shows a top-down view of a two-pixel device (i.e.,a device containing two sensing regions in the detector layer), wherethe ZMW/nanowell 702 is positioned above the border between the twosensing regions, 712 a and 712 b. It should be noted that theintervening diffractive beam shaping element, color filtration layer,and other features of the device are omitted from the view of FIG. 7A.FIG. 7B shows a side view of the same device, indicating how lightemitted from the ZMW/nanowell would pass through diffractive beamshaping element 708 and color filtration elements 710 a and 710 b toreach sensing regions 712 a and 712 b of the detector layer.

The design of the diffractive beam shaping elements of the instantdevices can be varied as desired to obtain the desired spatialseparation, collimation, and/or focusing of emission light passingthrough the element. For example, as shown in FIGS. 8A-8C, a nominaldesign (panel A) can include sufficient space between the diffractivebeam shaping element and the detector layer to allow the inclusion of,for example, a laser rejection interference filter layer (see below) orother optical feature. In some situations, it can be advantageous toincrease the lateral spacing between the diffractive beam shapingelement and the detector layer (panel B), whereas in other situations,it can be advantageous to build a more compact structure, by decreasingthe lateral spacing between the diffractive beam shaping element and thedetector layer (panel C). The alterations in optical properties of thediffractive beam shaping element are readily achieved by modification ofthe design of the diffractive beam shaping element, as would beunderstood by those of ordinary skill in the art.

FIGS. 9A-9B demonstrate the simulated transmission of emission lightfrom the emission volume of a ZMW/nanowell through a representativediffractive beam shaping element design. The basic design of thesimulated device is shown schematically in FIG. 9A, including thepositions of the ZMW/nanowell 902, the diffractive beam shaping element908, and the color filtration layer 910. As shown in FIG. 9B, theintensity of light transmitted through a diffractive beam shapingelement designed as shown in FIG. 9A is spatially separated by thediffractive effects of the diffractive beam shaping element. Thecomposition and structure of diffractive beam shaping element 908, asshown in FIG. 9A, were designed using ZEMAX optical ray tracingsoftware, and the transmission properties of light through thediffractive beam shaping element, as shown in FIG. 9B, were modeledusing Lumerical FDTD (finite difference time domain) Maxwell equationselectro-magnetic propagation software.

It should be noted that the instant diffractive beam shaping elementsare not intended to separate light based on color. Rather, it is afeature of these diffractive beam shaping elements that they providemaximal efficiency of transmission of all spectra, and that colordistinction is provided by the color filtration layer. In this regard,FIGS. 10A-10D show the efficiency and effect of wavelength on emissionpassage through a twin diffractive beam shaping element in the absenceof color filters. The phase pattern of the diffractive beam shapingelement, as viewed from above, is shown in FIG. 10A. The field profileat the detector level for a 630 nm emission is shown in FIG. 10B. Theefficiency of the diffractive beam shaping element as a function ofemission wavelength is shown in FIG. 10C for a collection path withoutany lens patterning but with the a-Si deposited. The 2 μm oxide line isfor a device with the detector 2 μm away from the a-Si layer, and the 4μm oxide line is for a device with the detector 4 μm from the a-Silayer. In these devices, the pixel is relatively large (˜8 μm×10 μm).The effect of wavelength on focus of the device is shown in FIG. 10D.

FIGS. 11A-11B illustrate the design, construction, and analysis of ananoscale device fabricated according to the instant disclosure,including a diffractive beam shaping element for spatial separation oflight transmitted from an emission volume. FIG. 11A shows the designfeatures of the device, including the ZMW/nanowell 1102, the diffractivebeam shaping element 1108, and the color filtration layer 1110. Thedevice further includes a waveguide (WG) for delivery of excitationlight to the ZMW/nanowell, metallic (Al) and antireflective (TiN)coatings on the surface of the device, silicon oxide spacer layers aboveand below the diffractive beam shaping element, and an aperture layer onthe surface of the diffractive beam shaping element. Approximatedimensions of the various features are provided in the drawing. FIG. 11Bshows an SEM micrograph corresponding to the device constructedaccording to the design outlined in FIG. 11A.

Aperture Layers

The integrated analytical devices of the instant disclosure canoptionally include one or more aperture layers. The aperture layers arefabricated between other layers of the nanoscale analytical devices, forexample between the ZMW/nanowell layer and the diffractive beam shapingelement layer, between the diffractive beam shaping element layer andthe color filtration layer, and/or between the color filtration layerand the detector layer. The apertures provide openings to allow maximumtransmission of emitted light from the ZMW/nanowell to the sensingregions of the detector element within a given unit cell, while at thesame time minimizing background transmission of light, either from theexcitation source (e.g., the waveguide), from autofluorescence withinthe device, or from cross-talk between adjacent unit cells. Aperturelayers are typically constructed of light-blocking materials wheretransmission of light is undesirable and of transparent materials wheretransmission of light is desired. Suitable light-blocking materials foruse in the aperture layers include, for example, titanium nitride,metals such as chromium, or any other appropriate light-blockingmaterial. The light-blocking material is preferably titanium nitride.Suitable transparent materials for use in the aperture layers include,for example, SiO₂, Si₃N₄, Al₂O₃, TiO₂, GaP, and the like. In preferredembodiments, the aperture layer is approximately 100 nm thick.

Laser Rejection Filter Elements and Color Filtration Elements

The integrated analytical devices of the instant disclosure additionallyinclude features designed to transmit certain wavelengths of light,while significantly decreasing or blocking other wavelengths of light.In particular, it is desirable to transmit as much signal-related lightas possible to the appropriate region of the detector, and to block all,or at least most, noise-related light. Furthermore, since the lenselements of the instant devices are designed to transmit all wavelengthsof light emitted from an analyte, it is typically necessary to employcolor filtration elements between the lens elements and the differentsensing regions of the detector layer in order to distinguish differentemitters in the analyte.

The devices therefore include a color filtration layer disposed betweenthe lens element layer and the detector layer. A different colorfiltration element within the color filtration layer is typically usedfor each of the spatially-separated beams transmitted through the lenselement. The spatially-separated light typically passes through thecolor filtration layer before being detected by the correspondingsensing region in the detector layer. In some embodiments of theinvention, the color filtration layer comprises a plurality of colorfiltration elements, each color filtration element specific for a rangeof light wavelengths. In more specific embodiments, the color filtrationlayer comprises 2 to 9 color filtration elements. In even more specificembodiments, the color filtration layer comprises 2 color filtrationelements, sensing regions, and separated beams.

The devices can additionally and optionally include one or more laserrejection filter elements within a laser rejection filter layer. Thelaser rejection filter layer is disposed between the excitation sourceand the detector layer, typically between the color filtration layer andthe detector layer of the integrated devices. Such laser rejectionfilter elements (also known as “pump” rejection elements) are ofparticular importance in the case of fully integrated analyticaldevices, such as the devices of the instant disclosure, since theintegrated nature of these devices can place constraints on theaggregate thickness of all layers, and can also increase the angularbandwidth over which the rejection must be assured. For a non-integrateddetector device, the deposited layers responsible for rejection ofnon-signal light can be many tens of microns thick (summing over severalfilters participating), but typically only need to reject light over anangular range of <10 degrees (including both field of view (“FOV”) andfilter tilt). For integrated devices such as the devices exemplifiedherein, however, the layers for pump rejection may need to be as thin as5 microns or even less.

A further consideration with an integrated device is assuring that therejected, non-signal light be terminated effectively (i.e., that it beefficiently removed from the optical system, for example by convertingit to heat by absorption). For a non-integrated device, such terminationis generally not critical, whereas for an integrated device, thereflected light can reach another detector site with a few (inprinciple, one) reflections, and furthermore, there is no local exitport for the rejected light to escape from the device. For thesereasons, it is important to ensure that scattered light be converted toheat efficiently, ideally in one reflection. The detailed properties oftwo types of laser rejection filter elements suitable for use in theinstant integrated devices is described in subsequent sections of thedisclosure.

The color filtration elements and the laser rejection filter elementshave features in common with one another, in that they are both designedto transmit certain wavelengths of light while blocking otherwavelengths of light. The color filtration elements, however, serve todistinguish between wavelengths of light emitted from different emittersin the analyte, whereas the laser rejection filter elements are designedto block background noise arising from the waveguide or other excitationsources by scattering or other means. Accordingly, different colorfiltration elements are typically placed between the spatially-separatedlight from the lens element and the plurality of sensing regions in thedetector layer, and a single laser rejection filter element—or multiplelaser rejection filter elements with similar properties—is typicallyplaced between the lens element and the detector layer, preferablybetween the color filtration layer and the detector layer. Suitablematerials for use in the color filtration and laser rejection filterelements of the instant devices include, for example, amorphoussilicon/silicon oxide interference stacks, polymer-like resists, dopedPECVD oxides, organo-silicone with absorbing dyes, and the like. Inpreferred embodiments, the color filtration and laser rejection filterelements are thin-film interference filters. In more preferredembodiments, the color filtration and laser rejection filter elementsare prepared from layers of amorphous silicon and silicon oxide. Inother preferred embodiments, the laser rejection element is disposedbetween the color filtration layer and the detector layer.

Multilayer and Hybrid Laser Rejection Filter Elements

An ideal laser rejection filter provides for the deep rejection ofoptical energy at the wavelengths of sample excitation (e.g., OD>=6 at532 nm for a typical laser illumination source), displays a broad windowof high transmission at the wavelengths of sample emission, and furtherdisplays a small Stokes shift between the wavelengths of interest. Inaddition, it is desirable for a laser rejection filter to displayminimal dispersion with angle and polarization, minimal thickness, andcontrolled termination. Furthermore, the filter stacks are preferablyinexpensive and readily manufacturable under conditions (e.g.,temperatures) suitable for the manufacture of other components of anintegrated device.

In the case of dielectric thin-film laser rejection filters, it cansometimes be challenging in the design of such stacks to obtain adequatefilter performance over a wide range of incident angles for thenon-signal light. For example, given a specified wavelength range, anedge filter can provide high reflection efficiency but only within aparticular range of incident angles (typically from normal incidence upto a certain value). In some of the integrated device designs describedherein, in order to keep the scattering photons of the excitation sourcefrom reaching the detector, rejection over a wide angular spectrum maybe desirable, especially to block photons with higher angle of incidencethan a typical thin film stack can adequately support.

The instant disclosure addresses this problem by providing in one aspectmultilayer laser rejection filters comprising a low index total internalreflection (TIR) layer in order to reduce transmission of high anglescattering light. Specifically, the low index layer is included in thedevice stack between the excitation source and the detector layer inorder to minimize the background signal. Traditional dielectriclong-pass filters, for example as shown in the left panel of FIG. 12A,reflect rays with lower angles of incidence (e.g., the middle rays inthe drawing) more effectively than those with higher angle of incidence(e.g., the outer ray in the drawing). As shown in the right panel ofFIG. 12A, when this filter design is incorporated into an integrateddevice, the high angle scattering light from the waveguide has arelatively higher chance of being transmitted through the filter stackand reaching the sensor. In the design solution of the instantdisclosure, however, for example in the structure shown in the leftpanel of FIG. 12B, a low index TIR layer is added between the integratedexcitation waveguide and a low angle rejection filter, such as adielectric filter stack. The high angle scattering light experiencestotal internal reflection upon encountering the low index TIR layer, andafter multiple bounces, exits the integrated device from the side. Atthe same time, the lower angle scattering light is transmitted throughthe low index TIR layer but is rejected by the dielectric filter stack.The combined effect of the TIR layer and the filter stack thus resultsin a barrier filter that blocks the scattering light with wide angularspectrum.

One candidate material for the low index TIR layer of the subjectmultilayer filter stack is air, with almost zero dispersion and lowrefractive index, but other low index materials are also suitable,including other gases, liquids, and solids having low refractive indexand other suitable properties. The specific choice of material for thelow index TIR layer will depend on the refractive index and otherphysical properties of the adjacent layers, as would be understood bythose of ordinary skill in the art.

To help collect the scattered light and reduce the chance of multiplescattering, an absorption layer or patch can optionally be added to thedevice, as shown in the right panel of FIG. 12B. Materials for use insuch an absorption layer are chosen based on their wavelength ofabsorption, their ability to dissipate optical energy, and theirsuitability in fabrication of the integrated device.

A variety of configurations of the above-described wide angular spectrummultilayer edge filter are possible, depending on the location,thickness, material choice, and number of layers of the low indexlayer(s). As described above, the low index layer can be placed directlybelow the excitation waveguide cladding, thus creating the shortestresonance cavity length and therefore limiting the chances for secondaryscattering. The low index layer may, however, alternatively be placedwithin the thin film stack, as illustrated in the left panel of FIG.12C, or between the thin film stack and the detection layer, asillustrated in the right panel of FIG. 12C. These configurationsincrease the resonance cavity length, and can therefore increase thechance of secondary scattering, but the configurations canadvantageously facilitate manufacture of the device. Not shown in theseexamples is the lens element layer, which could be either above or belowthe laser rejection filter element, but which is preferably above thelaser rejection filter.

In any case, incorporation of an additional TIR design constraint intothe laser rejection filter design generates added value to the low indexlayer. For example, by incorporating the low index layer (or layers) asan integral component in the laser rejection filter design, e.g.,because the filter is no longer limited to the thin film stack but caninclude the layers from the excitation waveguide to the detection layer,the integrated device performance can be fully optimized.

The instant disclosure further provides in another aspect laserrejection filter elements comprising a combination of dielectric stacksand absorption layers. Such hybrid filters take advantage of thecomplementary dependence on angle of incidence of interference coatingsand absorption layers. Specifically, as mentioned above, interferencecoatings for rejection typically perform best for a cone centered onnormal incidence, with dispersions that affect performance as a cosineof the angle in the interference thin films, whereas the performance ofabsorption rejection layers tends to increase with the angle ofincidence, with dispersions that affect performance as a cosine of theangle in the absorbing layer. Owing to this complementary nature, ahybrid coating can be achieved with rejection of a target minimum over awide angle range, in a minimum thickness. This thickness is reduced forhigher refractive index thin films, and for lower refractive indexabsorbing layers. Note that thin films with absorption for thenon-signal light (but minimal absorption of signal light) can be usedeffectively in a hybrid rejection filter.

As an example of an absorption dye suitable for use in combination witha dielectric filter stack, Aptina red1 has an absorption spectrum withhigh transmission above 600 nm. See Pang et al. (2011) Lab Chip 11:3698,FIG. 2. Although the thickness used in this publication was relativelylarge (8 μm), thinner layers can be used depending on the wavelength oflaser excitation of the device. For example, a 5 μm layer provides OD>6at 532 nm, a 4.7 μm layer provides OD>6 at 540 nm, and a 2.8 μm layerprovides OD>6 at 562 nm. Other absorption dyes and pigments suitable foruse in the instant hybrid filter stacks are readily identifiable bythose of ordinary skill in the art.

In particular, laser rejection by an absorption dye layer, such as by alayer of Aptina red1 dye, advantageously displays no polarizationdispersion, weak angle dispersion, and controlled termination ofnon-signal light. In addition, angularly non-uniform scatter can allowfor further thinning of the absorption dye layer. If certain portions ofthe hemisphere have lower intensity non-signal light to be rejected, orif the intensity has known polarization dependence at some angles, thisinformation can be used to further reduce the hybrid rejection filterthickness (for a given rejection target). The disadvantages of anabsorption rejection filter, for example a layer of Aptina red1 dye,include a moderately large extinction coefficient, a relatively largethickness (5 μm), and the need to use sample dyes with a fairly largeStokes shift (532 nm to ˜620 nm). These disadvantages can be offset togreat extent, however, by the combination of an absorption layer with adielectric stack in the instant hybrid rejection filters. FIG. 13illustrates the weak angle dispersion (left panel) and lack ofpolarization dispersion (right panel) of an absorption dye layer.

With respect to the dielectric stack component of a hybrid rejectionfilter, particularly advantageous rejection filters (especially thosewith low dependence on angle) are possible through the use of very highindex materials for the interference portion of the filter. Exemplarymaterials finding utility for these purposes with 532 nm pumps are GaP(gallium phosphide) as the high index material, and TiO₂ as the lowindex material, although other suitable materials could be utilized, asdescribed below, and as would be understood by those of ordinary skillin the art. Of note is that TiO₂ is typically used as a high indexmaterial for commonly produced coatings. FIG. 14 illustrates theadvantageous properties of a n_(H)/n_(L) GaP/TiO₂ dielectric stack, inparticular the high extinction coefficient in the region of a 532 nmpump source (indicated by downward arrow), and a controllable Stokesshift. The material also displays, however, a significant angulardispersion (with a blue shift) between 0 and 45 degrees, and asignificant polarization dispersion (splitting) between a p-polarizedoptical signal (upper trace near 570 nm) and an s-polarized opticalsignal (middle trace near 570 nm).

The advantages of combining an absorption dye layer and a dielectricinterference stack in a single hybrid laser rejection filter areillustrated in FIGS. 15A-15C. Specifically, FIG. 15A shows an exemplaryschematic design of such a hybrid filter, where the lower layer is aGaP/TiO₂ thin film stack and the upper layer is Aptina red1 dye. Thehybrid filter achieves OD=6 rejection with a 3 μm total thickness, whereOD=2 is provided by the absorption layer and OD=4 is provided by theinterference layer. Polarization dispersion and angular dispersion canbe compensated by the design of the filter element. As illustrated inFIGS. 15B and 15C, the effect of incident angle on transmittance isshown in FIG. 15B for p-polarized light (upper trace) and s-polarizedlight (lower trace), and the effect of wavelength on transmittance isshown in FIG. 15C for 45 degree incident light (left traces, p-polarizedand s-polarized) and for 0 degree incident light (right trace). Theabsorption layer controls termination of transmitted light, and theoverall design provides a tolerable Stokes shift, reasonable thickness,and good transmission at sample emission wavelengths.

The optical properties of the dielectric stack component of the hybridrejection filter can be modulated as desired by the choice of materialsused to construct the stack, by the thickness of each layer, and by thenumber of layers. The dielectric materials utilized to fabricateinterference filters are generally nonconductive materials, typicallymetal salts and metal oxides, having a specific refractive index.Exemplary materials include SiO₂, SiO, Si₂O₃, Al₂O₃, BeO, MgO, CeF₃,LiF, NaF, MgF₂, CaF₂, TiO₂, Ta₂O₅, ZrO₂, HfO₂, Sb₂O₃, Y₂O₃, CeO₂, PbCl₂,and ZnS. Also of use is GaP, due to its extremely high refractive index.The dielectric stack is preferably designed with overall structure (H/2L H/2)^(N), where the H layer is a first material with relatively highrefractive index and the L layer is a second material with relativelylow refractive index. The physical thickness of each layer within thestack is chosen based on the desired optical properties, as isunderstood in the art. The value “N” is the number of repeating units ofthe structure within the parentheses and is an integer. Transmission inthe stop band tends to zero (for a given incidence angle) withincreasing overall thickness (e.g., as N increases). FIG. 16Aillustrates the physical and optical properties of a GaP/TiO₂ stack withvarious values of N. FIG. 16B illustrates a further comparison of thephysical and optical properties of interference stacks using different Hand L pairs.

FIGS. 17A-17B and 18A-18B highlight the advantages of a GaP/TiO₂ stackin the hybrid rejection filter compared the use of other traditionaldielectric stack materials. The optical properties of a hybrid rejectionfilter comprising a GaP/TiO₂ dielectric stack and an Aptina red1absorption layer are described above and in FIGS. 15B and 15C. Forcomparison, the optical properties of a hybrid rejection filtercomprising a TiO₂/Al₂O₃ dielectric stack and an Aptina red1 absorptionlayer are shown in FIGS. 17A and 17B, and the optical properties of ahybrid rejection filter comprising a TiO₂/SiO₂ dielectric stack and anAptina red1 absorption layer are shown in FIGS. 18A and 18B.Importantly, the effective index of the TiO₂/Al₂O₃ and the TiO₂/SiO₂stacks are lower than that of the GaP/TiO₂ stack, thus resulting ingreater angle and polarization dispersion with these filters. Dispersioncompensation for the TiO₂/Al₂O₃ hybrid rejection filter requires a 4.7μm thickness (˜3.6 μm for the absorption layer and ˜1.1 μm for thedielectric stack). Dispersion compensation for the TiO₂/SiO₂ hybridrejection filter requires a 4.5 μm thickness (˜3.6 μm for the absorptionlayer and ˜0.9 μm for the dielectric stack). As is apparent from FIG.17B, the TiO₂/Al₂O₃ hybrid rejection filter would ideally be used withfluorescent dyes having relatively large Stokes shifts (e.g., 532 nmexcitation and >635 nm emission), and the TiO₂/SiO₂ hybrid rejectionfilter would be best used with dyes having even larger Stokes shifts.

It should be understood that the order of the coatings can be varied inorder to achieve optimal performance of the hybrid laser rejectionfilter elements. For example, the layers can be ordered with absorptionfirst, interference coatings second, or vice versa. The absorbingmaterial can be carried in a host material such as PMMA, and can beshaped or patterned to fit within limited volumes or to permit simplerintegration.

The coatings can be created in different process steps, and joined intoan assembly, as would be understood by those of ordinary skill in theart.

Accordingly, the instant disclosure thus provides in this aspect: Anarray of integrated analytical devices, each device comprising:

a nanoscale emission volume;

a detector layer optically coupled to the nanoscale emission volume;

a diffractive beam shaping element disposed between the nanoscaleemission volume and the detector layer;

a color filtration layer disposed between the diffractive beam shapingelement and the detector layer;

an excitation source optically coupled to the nanoscale emission volume;and

a laser rejection filter element disposed between the excitation sourceand the detector layer;

wherein light is emitted from the nanoscale emission volume by aplurality of emitters within the emission volume;

wherein the detector layer comprises a plurality of sensing regions; and

wherein the diffractive beam shaping element spatially separates thelight emitted from the nanoscale emission volume and directs thespatially-separated light through the color filtration layer to theplurality of sensing regions.

In some embodiments, the laser rejection filter element is a multilayeror a hybrid rejection filter element.

In specific embodiments, the laser rejection filter element is amultilayer filter element comprising a dielectric interference filterlayer and a low index total internal reflectance layer. In more specificembodiments, each of the devices further comprises an absorption layer.

In other specific embodiments, the laser rejection filter element is ahybrid rejection filter element comprising an absorption layer and adielectric stack layer.

In some embodiments, the laser rejection filter element displays lowoptical transmission at 532 nm and high optical transmission above 620nm.

Dark Mirror Elements

In another aspect, the integrated analytical devices of the instantdisclosure further comprise a dark mirror element. The term dark mirroris typically used to describe a surface with a coating that tends toabsorb incident light without inherently scattering the light and onethat also has low transmission. In integrated devices having a reservoirof non-sample fluorescent materials in the vicinity of the opticalsource, transmission of non-signal light into the reservoir offluorescent materials can result in added noise background and should beavoided. Placing dark mirror coatings on areas of the device notdirectly active in passing signal (or illumination) light improve theoverall ability of the integrated device to terminate non-signal lightefficiently before the rejected non-signal light can impinge on anotherdevice site.

The optical properties of exemplary dark mirror coatings are illustratedin FIGS. 19A-19B, where FIG. 19A shows that a significant reduction inreflectivity can be achieved on a dielectric stack coated with Cr. Withalternative coatings, for example a coating of TaN, even lowerreflectivity is possible, as illustrated in FIG. 19B. Other materialsare suitably used as dark mirror coatings, as would be understood bythose of ordinary skill in the art.

Dark mirror coatings can be placed on scattering surfaces to decreasethe probability of striking another device site within the signalangular bands, or to increase the path length for absorption beforereaching another device site.

Dark mirror coatings that are angle-sensitive and/orpolarization-sensitive can be used to permit highly efficienttransmission of signal light, while achieving some target level ofabsorption of non-signal light.

Accordingly, the instant disclosure thus provides in this aspect: Anarray of integrated analytical devices, each device comprising:

a nanoscale emission volume;

a detector layer optically coupled to the nanoscale emission volume;

a diffractive beam shaping element disposed between the nanoscaleemission volume and the detector layer;

a color filtration layer disposed between the diffractive beam shapingelement and the detector layer;

a dark mirror filter element;

wherein light is emitted from the nanoscale emission volume by aplurality of emitters within the emission volume;

wherein the detector layer comprises a plurality of sensing regions; and

wherein the diffractive beam shaping element spatially separates thelight emitted from the nanoscale emission volume and directs thespatially-separated light through the color filtration layer to theplurality of sensing regions.

In embodiments, the dark mirror element comprises a dark mirror coatingon a scattering surface.

Arrays of Integrated Analytical Devices

In order to obtain the volumes of sequence information that can bedesired for the widespread application of genetic sequencing, e.g., inresearch and diagnostics, high throughput systems are desired. As notedabove, and by way of example, in order to enhance the sequencingthroughput of the system, multiple complexes are typically monitored,where each complex is sequencing a separate template sequence. In thecase of genomic sequencing or sequencing of other large DNA components,these templates will typically comprise overlapping fragments of thegenomic DNA. By sequencing each fragment, one can then assemble acontiguous sequence from the overlapping sequence data from thefragments.

As described above, and as shown in FIGS. 1A-1B, the template/DNApolymerase-primer complex of such a sequencing system is provided,typically immobilized, within an optically confined region, such as azero mode waveguide (ZMW) or nanowell, or proximal to the surface of atransparent substrate, optical waveguide, or the like. Preferably, suchreaction cells are arrayed in large numbers upon a substrate in order toachieve the scale necessary for genomic or other large-scale DNAsequencing approaches. Such arrays preferably comprise a completeintegrated analytical device, such as, for example, the devices shown inthe block diagrams of FIGS. 2 and 5. Examples of integrated systemscomprising arrays of optical analytical devices are provided in U.S.Patent Application Publication Nos. 2012/0014837; 2012/0019828; and2012/0021525.

Arrays of integrated analytical devices, such as arrays of devicescomprising ZMWs/nanowells, can be fabricated at ultra-high density,providing anywhere from 1000 ZMWs per cm², to 1,000,000 ZMWs per cm², ormore. Thus, at any given time, it can be possible to analyze thereactions occurring in from 100, 1000, 3000, 5000, 10,000, 20,000,50,000, 100,000, 1 Million, 10 Million, or even more nanoscale emissionvolumes or other reaction regions within a single analytical system oreven on a single substrate.

Using the foregoing systems, simultaneous targeted illumination ofthousands or tens of thousands of ZMWs/nanowells in an array has beendescribed. However, as the desire for multiplex increases, the densityof ZMWs on an array, and the ability to provide targeted illumination ofsuch arrays, increases in difficulty, as issues of ZMW cross-talk(signals from neighboring ZMWs contaminating each other as they exit thearray), decreased signal:noise ratios arising from higher levels ofdenser illumination, and the like, increase. The arrays and methods ofthe instant invention address some of these issues.

The position on the detector upon which a given signal is incident isindicative of (1) the originating emission volume within a ZMW/nanowellin the array, and (2) the emission characteristics of the signalcomponent, which is used, for example, to identify the type offluorescently labeled nucleotide analog incorporated in an extensionreaction. As noted above, the detector can include in some cases aplurality of sensing regions, each for detecting light passed from theemission volume through a diffractive beam shaping element and a colorfiltration layer to a detector layer. For example, in the case ofsequencing, the sensor for each reaction cell can have 4 elements, onefor each of the four bases. In some cases, the sensing regions canprovide color discrimination, although the color filtration layer ispreferably used to distinguish the appropriate color of light for theappropriate sensing region. In these embodiments, the sensing regionsdetect intensity of signal only, without discriminating color. In somecases, the sensor elements identify the incorporated nucleotide using acombination of emission characteristics.

FIGS. 20A-20D illustrate exemplary device layouts usefully employed inthe arrays of the instant disclosure. In each case, the arrays areviewed from above, with dark circles representing the ZMWs/nanowells. Asshown, the ZMWs/nanowells are positioned directly above waveguides,which are identified as broad arrows. In the case of the arrays shown inFIGS. 20A and 20C, the “pitch” of the waveguide is 2 columns (i.e., thewaveguides are separated by the width of two columns of sensingregions/pixels), whereas for the arrays of FIGS. 20B and 20D, the pitchof the waveguide is 1 column (i.e., the waveguides are separated by thewidth of one column of sensing regions/pixels). The spatial separationof emitted light effected by the diffractive beam shaping elements ineach of the arrays is indicated by the two thin arrows associated withsome of the ZMW/nanowells. For example, in the devices of FIG. 20A, thediffractive beam shaping elements direct emitted light onto the twosensing regions that are aligned perpendicularly (i.e., at 90°) to thewaveguide. In the devices of FIG. 20B, the diffractive beam shapingelements direct emitted light onto the two sensing regions that arecollinear (i.e., at 0°) with the waveguide. For the devices of FIGS. 20Cand 20D, the diffractive beam shaping elements direct emitted light ontothe two sensing regions that are diagonal (i.e., at 45°) relative to thewaveguide. As is apparent from the drawings, the devices of FIGS. 20Cand 20D differ with respect to the pitch of the respective waveguides.

FIG. 21 illustrates an array of devices, as viewed from above, whereinthe design of an exemplary diffractive beam shaping element 2108 withina specific unit cell is shown in more detail. Also labeled within theunit cell is a ZMW/nanowell 2102, a waveguide 2105, and one of the twocolor filtration regions 2110 that would be positioned over a sensingregion of the detection layer. In these devices, the sensing regionswould be collinear with the waveguide, and the waveguide pitch would be1 column. Not shown in this drawing is the second color filtrationregion and various other features of the devices that have beendescribed above, e.g., aperture elements, laser rejection elements,metallic and anti-reflective surface layers, waveguide cladding layers,electronic circuitry, and so forth.

FIGS. 22A-22E show the various typical elements used to build anexemplary unit cell of the instant devices and their general features.The elements are viewed from above the plane of the unit cell.Specifically, FIG. 22A illustrates a diffractive beam shaping element,where the dimensions of the diffractive beam shaping element (and alsothe unit cell itself) are roughly 10 μm×15 μm. FIG. 22B illustrates aZMW/nanowell (shown as a small square) and its associated waveguide. Asindicated, the width of the waveguide is roughly 0.3 to 0.7 μm, and itis fabricated in a trench of approximately 9 μm wide. FIG. 22Cillustrates two circular color filtration regions of radius 2.8 μm and3.0 μm. As assembled within the device, however, each of the colorfiltration regions would be offset approximately 3.0 μm relative to theZMW/nanowell. FIG. 22D illustrates a ZMW/nanowell (shown as a smallsquare) and two associated aperture elements, which correspond in eachcase to two offset transparent circles. The larger aperture elementlayer, with circles having diameters of approximately 2.5 μm, and offsetapproximately +/−2.7 μm relative to the ZMW/nanowell, would typically bedisposed between the detector layer and the laser rejection layer/colorfiltration layer. The smaller aperture element layer, with circleshaving diameters of approximately 2.0 μm, and offset approximately+/−1.6 μm relative to the ZMW/nanowell, would typically be disposedbetween the color filtration layer and the diffractive beam shapingelement layer. FIG. 22E illustrates a ZMW/nanowell (shown as a smallsquare) and a third associated aperture element, corresponding to twooffset transparent circles. These circles have diameters ofapproximately 1.5 μm, and are offset approximately +/−1.0 μm relative tothe ZMW/nanowell. This aperture element would typically be disposedbetween the diffractive beam shaping element layer and the waveguide.

FIG. 23 illustrates a schematic representation of the assembly of thecomponents of FIGS. 22A-22E into an exemplary integrated unit celldevice of the invention. The ZMW/nanowell is apparent as a small squarein the center of the unit cell, and the waveguide is shown as thevertical parallel lines straddling the ZMW/nanowell. The exemplarydevice includes from top to bottom, in the following order, theZMW/nanowell, the waveguide, a third aperture element, a diffractivebeam shaping element, a second aperture element, a color filtrationlayer, a first aperture element, and a detector layer. The exemplarydevice can optionally include a laser rejection layer between the colorfiltration layer and the first aperture element, or at another locationin the device. The diffractive beam shaping element in this embodimentof the nanoscale integrated analytical device would direct light emittedfrom the ZMW/nanowell perpendicular to the waveguide. In other words,this exemplary device would correspond to the array layout shown in FIG.20A.

FIG. 24 provides a more detailed schematic cross-section of the devicedescribed in FIG. 23, including dimensions and exemplary materials. Across-sectional SEM micrograph of a device fabricated according to thedesign of FIG. 24 is shown in FIG. 25.

Methods for Producing Arrays of Integrated Analytical Devices

In another aspect, the instant disclosure provides methods for producingarrays of integrated analytical devices. As described above, such arraysare useful, for example, in the large-scale sequencing of nucleic acids,including in particular, genomic sequencing. Such arrays can be producedby a variety of methods. One preferred approach for producing theinstant arrays involves the use of microfabrication methods such assemiconductor or MEMS processing methods, which have been highlydeveloped for the production of integrated circuits. Similar processeshave been used to create MEMS (micro electromechanical systems) for avariety of applications including inkjet printers, accelerometers,pressure transducers, and displays (such as digital micromirror displays(DMDs)). Microfabrication methods can be applied to a large substratesuch as a wafer, which can later be diced into many devices, allowingfor the production of many devices at one time.

The methods of the invention may, for example, apply resist processes,such as photoresists, to define structural elements on substrates orother layers. Etching processes can be used to produce three-dimensionalstructures, including component structures in the integrated analyticaldevice. Deposition processes can be used to add layers onto the devices.Other semiconductor processes such as ashing, polishing, release,liftoff, and wet cleans can also be employed to create the structures ofthe invention, as described in more detail below.

For example, lithographic techniques can be used to define a mask layerout of polymeric materials, such as photoresists, using e.g.,conventional photolithography, e-beam lithography, or the like.Alternatively, lithographic techniques can be applied in conjunctionwith layer deposition methods to deposit metal mask layers, e.g., usingaluminum, gold, platinum, chrome, or other conventionally used metals,or other inorganic mask layers, e.g., silica based substrates such assilicon, SiO₂, or the like. Alternatively, negative tone processes canbe employed to define pillars of resists that correspond to, forexample, nanowells. See, e.g., U.S. Pat. No. 7,170,050, which isincorporated by reference herein in its entirety for all purposes. Themask layer can then be deposited over the resist pillars and the pillarsare subsequently removed. In particularly preferred aspects, both theunderlying substrate and the mask layer are fabricated from the samematerial, which in particularly preferred aspects, is a transparentsubstrate material such as an SiO₂-based substrate such as glass,quartz, or fused silica. By providing the mask and underlying layers ofthe same material, one can ensure that the two layers have the sameinteractivity with the environments to which they are exposed, and thusminimize any hybrid surface interactions.

In the case of SiO₂-based substrates and mask layers, conventionalfabrication processes can be employed. For example, a glass substratebearing a surface-exposed feature, such as a waveguide, can have a layerof resist deposited over its surface. A negative of the mask layer isthen defined by appropriate exposure and development of the resist layerto provide resist islands where one wishes to retain access to theunderlying feature. The mask layer is then deposited over the surfaceand the remaining resist islands are removed, e.g., through a lift offprocess, to provide the openings to the underlying feature. In the caseof metal layers, deposition can be accomplished through a number ofmeans, including evaporation, sputtering or the like. Such processes aredescribed in, e.g., U.S. Pat. No. 7,170,050. In the case of silica basedmask layers, a chemical vapor deposition (CVD) process can be employedto deposit a silicon layer onto the surface. Following lift off of theresist layer, a thermal oxidation process can convert the mask layer toSiO₂. Alternatively, etching methods can be used to etch access pointsto underlying layers using conventional processes. For example, asilicon layer can be deposited over an underlying substrate. A resistlayer is then deposited over the surface of the silicon layer andexposed and developed to define the pattern of the mask. The accesspoints are then etched from the silicon layer using an appropriatedifferential etch to remove silicon but not the underlying SiO₂substrate. Once the mask layer is defined, the silicon layer is againconverted to SiO₂ using, e.g., a thermal oxidation process.

One aspect of the invention relates to a process for producing arrays ofintegrated analytical devices comprising the steps of: providing asubstrate layer, which can be a light-sensitive detector layer, such asa CMOS sensor layer, a CCD layer, or the like; depositing a laserrejection filter element layer on the substrate layer; depositing acolor filtration layer on the laser rejection filter element layer;depositing a lens element layer, specifically a layer including adiffractive beam shaping element, on the color filtration layer;depositing an excitation waveguide layer on the lens element layer,depositing a ZMW layer on the lens element layer; and patterning andetching the ZMW material to define an array of nanowells penetratinginto the upper cladding of the ZMW layer. Unless specifically described,the order of the steps of the processes described herein can be altered,where suitable. In some embodiments, additional steps can be added, inparticular the deposition and patterning of one or more aperture layersbetween the other layers of the device. A specific example of such afabrication process is provided in detail below. Further examples ofprocesses useful in the production of arrays of integrated analyticaldevices can be found in U.S. patent application Ser. No. 13/920,037,which is incorporated by reference herein in its entirety for allpurposes.

In each of the above exemplary microfabrication techniques, the processbegins with a clean substrate layer. The substrate layer used in theinstant methods can be of any suitable rigid material. The substratelayer material can comprise, for example, an inorganic oxide materialsuch as silica. A preferred substrate layer material comprises adetector layer, such as, for example, a CMOS wafer, i.e., a wafer madeup of CMOS sensors or CCD arrays. See, for example, CMOS Imagers FromPhototransduction to Image Processing (2004) Yadid-Pecht andEtienne-Cummings, eds.; Springer; CMOS/CCD Sensors and Camera Systems(2007) Holst and Lomheim; SPIE Press.

As mentioned above, the methods of the invention in some cases useresists for defining and producing structures with lithography. Theseresists can be, for example, photoresists or e-beam resists. Thephotoresists can be developed using UV, deep UV, G-line, H-line, Mine orother suitable wavelength or set of wavelengths. The type of resist thatis used, and therefore the type of instrumentation that is employed forprocessing, will depend on the dimensions of the features that arecreated. In many processes described herein, higher resolution resistsand equipment will be used for the production of the nanowell whichcorresponds to the reaction volume, where the size of the nanowell canbe on the order of 10 nm to 500 nm, and a lower resolution resist andassociated instrumentation is used for the creation of the rest of theintegrated analytical device, which can have features on the dimensionsof 1 micron to 20 microns. Many resists are known in the art, and manyare available commercially from companies such as Rohm and Haas andShipley. The resists used in the processes of the invention can benegative or positive photoresists. Where a process is described hereinusing a negative photoresist, it is to be understood that a suitablepositive photoresist can also be employed where practical, and viceversa. Where appropriate, chemical amplification can also be employed inorder to increase the sensitivity of the resist. The removal of theresist, the cleaning, rinsing, ashing, and drying of the substrate canbe performed as appropriate and as taught and known in the art.

In some cases, the tools used for photolithography of the nanowell usephotolithography exposure tool capable of creating structures havingfeature sizes of about of 10 nm to about 100 nm. Such systems include,for example, an AMSL XT1250 exposure tool.

Etching processes are used in some aspects of the invention in order toproduce the three dimensional features in a substrate or in otherlayers, to fashion, for example, optical elements or lenses, or reactionvolumes such as nanowells. The etching process that is used will dependon the type of material used, the dimensions of the features, and theresist system. In some cases wet etching or wet chemical etching isemployed. Electrochemical etching can also be employed. In someembodiments plasma etching or reactive ion etching (RIE) is used as anetching process. Deep reactive ion etching (DRIE) can also be employed,for example, where structures having high aspect ratio are desired. Dryvapor phase etching, for example with xenon difluoride, can also beused. Bulk micromachining or surface micromachining can be used asappropriate to create the device structures of the disclosure. Theetching used in the methods of the disclosure can be gray-scale etching.The conditions of the resist formation and etching are controlled toproduce side walls having the desired geometries, such as having thedesired side-wall angle.

Some processes of the invention involve the deposition of reflectivelayers, or cladding layers. The deposition of these reflective layerscan be accomplished by wet processes including spinning on layers fromsolution, or by gas-phase processes. Suitable processes includeelectroplating, sputter deposition, physical vapor deposition,evaporation, molecular beam epitaxy, atomic layer deposition, andchemical vapor deposition. Metals can be used as the reflective layerand the cladding layer. Suitable metals include gold, nickel, aluminum,chromium, titanium, platinum, and silver. The reflective and/or claddinglayers can comprise aluminum, which can be deposited by sputtering, forexample using a commercially available sputter tool available from CVC,Novellus, or MRC.

Where layers are deposited during the processes of the invention, insome cases, the layers are treated before moving on to the next step inthe process. For example, the deposited layer can be annealed,planarized, cleaned, passivated, or lightly etched in order to improveits properties.

In some methods of the invention, protective layers or sacrificiallayers are deposited. The protective layers can be polymeric layers, orcan be inorganic layers. Suitable protective or sacrificial layersinclude germanium (Ge) and amorphous silicon (a-Si). Protective layerscan be used to produce features as described herein. The type ofmaterial for the protective or sacrificial layer can be chosen for itsselective reactivity, for example to wet chemical etchants. For example,in some cases, the ability to selectively etch germanium with heatedhydrogen peroxide in the presence of silicon dioxide and aluminumresults in its being utilized to produce optical structures combinedwith nanowells.

In some processes, a pull-back process can be employed. A pull-backprocess generally involves etching in from the edges of a feature withina layer in order to reduce the dimensions of the feature. Pull-back canbe performed using a wet chemical reagent that selectively reacts with alayer which has exposed edges. In some cases a germanium layer is pulledback using hydrogen peroxide.

Some methods employ a polishing step to remove a surface region from asurface. Suitable methods include chemical-mechanical polishing orchemical-mechanical planarization (CMP).

Some methods of the invention incorporate a planarization layer. Themethod for depositing the planarization layer depends on the type ofmaterial that is used. The planarization layer can be a hard material,such as an inorganic material, for example silicon nitride; it can be ametallic material such as aluminum; or it can be a soft material, suchas a polymeric material, e.g. an organic or silicon based polymer. Theplanarization layer can be a glass, such as a silicon dioxide material.In some cases, the planarization layer comprises a spin-on glass such asa silicate, phosphosilicate or siloxane material. Suitable spin-on glassmaterials are available, for example, from Honeywell Corporation. Theplanarization layer can comprise, for example, a glass doped with otheragents to control its melting properties, such a boro-phosphoro-silicateglass (BPSG). Suitable polymeric planarization materials include, forexample, polyimides.

After the arrays of the instant disclosure are complete, such as by, forexample, following the process flow of the example below, the arrays canbe further processed, such as, for example, by separating the arraysinto individual chips and readying them for sequencing. The furtherprocessing steps will depend on the situation but can typically includethe following treatments: surface treatment (a series of wet/vapor phasetreatments to put down a specific surface that attracts the DNApolymerase enzyme to the bottom of the nanowell); stacking (a process toprotect the top surface of the surface-treated device wafer and, in somecases, creating a well for the sequencing mixture); thinning (a processin which the composite top-plated and surface-treated device wafer canbe thinned—including grinding lapping, polishing, or other treatments);dicing (a process in which the composite wafer is divided intoindividual chips using a standard semiconductor dicing saw); andpackaging (a process involving a standard pick and place tool to mountthe chips onto a substrate and create electrical/optical outputs fordata collection). These further processing steps are either known in theart or are disclosed in references such as U.S. Patent ApplicationPublication Nos. 2008/0176769 and 2011/0183409, which are incorporatedby reference herein in their entireties for all purposes.

As just noted, the arrays of the invention can be incorporated intoanalysis systems for analyzing the multiple reactions occurring in thenanowells of the array. The arrays described herein typically havenanowells that are accessible to fluid from the top, and that areaccessible for optical analysis from the bottom. The arrays are thusgenerally incorporated into a vessel into which a reaction mixture ofinterest is introduced. In some cases, the individual nanowells are allin contact with one volume of fluid, which can have, for example,multiple nucleic acid template molecules which can be analyzed, andwhich can have the nucleotides, cofactors, and other additives forcarrying out the reaction to be analyzed.

The vessel that comprises the array can be placed within an instrumentwhich has the appropriate optical components, computer controls, anddata analysis systems. The vessel comprising the array can be heldwithin the instrument such that the reaction conditions, such as thevessel temperature and vessel atmospheric conditions, can be controlled.The vessel atmospheric conditions can comprise the makeup of the gasabove the sample, for example the humidity, and the level of othergaseous species such as oxygen.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein can be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following Example, which is includedherewith for purposes of illustration only and is not intended to belimiting of the invention.

EXAMPLE

An exemplary semiconductor fabrication process according to one aspectof the instant invention is shown in FIGS. 26A-26R, which illustratefabrication of an array of integrated analytical devices comprising adiffractive beam shaping element to spatially separate light emittedfrom a nanoscale emission volume and direct the spatially-separatedlight through a plurality of color filters to a plurality of sensingregions within a detector layer.

In the exemplary methods, the process begins with a clean semiconductorsubstrate layer, preferably an integrated CMOS detector layer, althoughthe array could be designed to be attached to a separate detectordevice. Accordingly, the substrate layer can be of any suitable rigidmaterial with sufficient transparency to light emitted from the reactionwell. For examples of appropriate detector layers, see CMOS Imagers FromPhototransduction to Image Processing (2004) Yadid-Pecht andEtienne-Cummings, eds.; Springer; CMOS/CCD Sensors and Camera Systems(2007) Holst and Lomheim; SPIE Press.

The surface of the substrate can be prepared for deposition by, forexample, a wet strip process or other suitable cleaning step. Bond padsare opened through passivation of the SiN layer, and the surface isfilled with oxide and planarized as shown in steps 1-3 of FIG. 26A.

The sensor substrate layer is next patterned with oxide to generatepattern zero layer alignment marks, as shown in steps 4-6 of FIG. 26A.The patterning aligns to CMOS top metal alignment marks, and zero marksthe lowest level alignment layer used for subsequent stack patterning.Aperture 1 is deposited as shown in steps 7-12 of FIG. 26A. As noted inmore detail above, this layer is used to screen out low angle signalnoise from the diffractive beam shaping element. In addition, theaperture 1 layer can also decorate zero marks to make it more easilyvisible for subsequent alignments. In this example, the aperture layeris composed of titanium nitride and is approximately 100 nm thick. Agraphical representation of the sensor substrate and aperture 1 layer isshown in FIG. 26B.

The laser rejection filter layer is next deposited, as shown in steps13-15 of FIG. 26C. The filter comprises alternating layers of amorphoussilicon and silicon dioxide, deposited as shown. A graphicalrepresentation of the sensor substrate, aperture 1 layer, and laserrejection layer is shown in FIG. 26D. Note that step 14 also includesdeposition of CF1, the first portion of the color filtration layer. Thislayer is not shown in FIG. 26D.

The color filtration layer is prepared on top of the laser rejectionfilter, as shown in steps 16-26 of FIG. 26E. For this example, there aretwo separate sensing regions on the detector layer for each device ofthe array, so the color filtration layer in each device comprises twodifferent color filters, CF1 and CF2, as shown graphically in FIG. 26F.The filters themselves comprise alternating layers of amorphous siliconand silicon dioxide, deposited as shown. Color filter CF1 is depositedas part of the laser rejection filter deposition. It is patterned andetched in steps 16-19 of the process. Color filter CF2 is deposited instep 20 of the process, and is patterned and etched in steps 22-25. Agraphical representation of the sensor substrate, aperture 1 layer,laser rejection filter layer, and color filtration layer is shown inFIG. 26F. In this example, filters CF1 and CF2 differ only in 2 oxidelayers in thickness.

The second aperture layer in this example, aperture 2, is prepared asshown in steps 27-32 of FIG. 26G, and the lens spacer oxide layer isprepared on top of this layer as shown in steps 33-36. A graphicalrepresentation of the sensor substrate, aperture 1 layer, laserrejection layer, color filtration layer, aperture 2 layer, and lensspacer oxide layer is shown in FIG. 26H.

The lens element layer, which comprises a diffractive beam shapingelement, is prepared by lithographic patterning of the lens space oxidelayer, as shown in steps 37-46 of FIG. 26I, and then deposition of acarbon-rich amorphous silicon and polishing, as shown in steps 47-48 ofFIG. 261. A graphical illustration of the intermediate substrate formedafter lithographic patterning of the lens spacer oxide layer and beforedeposition of the carbon-rich amorphous silicon is provided in FIG. 26J.

The patterned, filled, and polished lens layer is next patterned,deposited with titanium nitride, and patterned again to form aperture 3,as illustrated in steps 49-56 of FIG. 26K. The resultant substrate,which includes sensor substrate, aperture 1 layer, laser rejectionfilter layer, color filtration layer, aperture 2 layer, lens spaceroxide layer, lens layer, and aperture 3 layer is illustrated graphicallyin FIG. 26L.

Subsequent steps 57-68, as provided in FIG. 26M, represent thedeposition of a reflector oxide layer. The result of this deposition isillustrated in FIG. 26N.

An excitation waveguide layer is added to the substrate as shown steps69-78 of FIG. 26O. In this example, the material comprising the siliconnitride waveguide is deposited in step 69, the waveguide is etched insteps 71 and 74, and the oxide cladding is deposited in steps 76 and 78.The result of these process steps are illustrated graphically in FIG.26P, which includes sensor substrate, aperture 1 layer, laser rejectionfilter layer, color filtration layer, aperture 2 layer, lens spaceroxide layer, lens layer, aperture 3 layer, and the waveguide layer.

Fabrication of the zero-mode waveguide (ZMW) layer is shown in steps79-91 of FIG. 26Q. Specifically, this figure shows the deposition of thealuminum/titanium nitride surface layer in step 79, and the subsequentlithographic opening of the ZMW hole in step 86. The result of theaddition of this layer is illustrated graphically in FIG. 26R, whichincludes all of the above layers and also the ZMW/nanowell layer. Itshould be noted that the dimensions shown in any of the views of FIGS.26A-26R are for purposes of illustration only, and should not be takento be limiting in any way.

After all other process flow steps are complete, the arrays are treatedto remove all residues using a cleaning process step. Additional stepscan include, for example, deep etching steps to generate contacts to theCMOS bond pads and to couple the arrays to other components of thedevice.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined by reference to theappended claims, along with their full scope of equivalents.

1-91. (canceled)
 92. An array of integrated analytical devices, eachdevice comprising: a nanoscale emission volume; a detector layeroptically coupled to the nanoscale emission volume; an excitation sourceoptically coupled to the nanoscale emission volume; and a laserrejection filter layer disposed between the excitation source and thedetector layer; wherein light is emitted from the nanoscale emissionvolume by a plurality of emitters within the emission volume; whereinthe detector layer comprises a sensing region; wherein the laserrejection filter layer comprises a multilayer filter element comprisinga low index total internal reflectance layer; and wherein the low indextotal internal reflectance layer comprises a gas.
 93. The array of claim92, wherein the gas is air.
 94. The array of claim 92, wherein themultilayer filter element further comprises a dielectric interferencefilter layer.
 95. The array of claim 94, wherein the low index totalinternal reflectance layer is disposed between the excitation source andthe dielectric interference filter layer.
 96. The array of claim 94,wherein the low index total internal reflectance layer is disposedwithin the dielectric interference filter layer.
 97. The array of claim94, wherein the low index total internal reflectance layer is disposedbetween the dielectric interference filter layer and the detector layer.98. The array of claim 92, wherein the array further comprises anabsorption layer or patch.
 99. The array of claim 92, wherein thedetector layer comprises a plurality of sensing regions
 100. The arrayof claim 99, wherein the array further comprises a diffractive beamshaping element disposed between the excitation source and the detectorlayer.
 101. The array of claim 100, wherein the diffractive beam shapingelement comprises a Fresnel lens.
 102. The array of claim 100, whereinthe diffractive beam shaping element comprises amorphous silicon. 103.The array of claim 100, wherein the diffractive beam shaping elementcollimates the light emitted from the emission volume.
 104. The array ofclaim 100, wherein the array further comprises a color filtration layerdisposed between the diffractive beam shaping element and the detectorlayer.
 105. The array of claim 104, wherein the color filtration layercomprises a plurality of color filtration elements, each colorfiltration element optically coupled to one detector layer sensingregion and specific for a range of light wavelengths.
 106. The array ofclaim 105, wherein the diffractive beam shaping element spatiallyseparates the light emitted from the nano scale emission volume into aplurality of beams and directs the spatially-separated light beamsthrough the color filtration elements and onto the sensing regions. 107.The array of claim 106, wherein each device comprises two colorfiltration elements, two sensing regions, and two spatially-separatedlight beams.
 108. The array of claim 106, wherein each device comprisestwo emitters within the emission volume.
 109. The array of claim 92,wherein the array further comprises at least one aperture layer disposedbetween the excitation source and the detector layer.
 110. The array ofclaim 109, wherein the aperture layer comprises titanium nitride. 111.The array of claim 92, wherein the excitation source is a waveguideexcitation source.
 112. The array of claim 111, wherein the nanoscaleemission volume is aligned directly above the waveguide excitationsource.
 113. The array of claim 92, wherein the detector layer isintegral to the device.
 114. The array of claim 92, wherein the detectorlayer is not integral to the device.
 115. The array of claim 92, whereinthe sensing region is rectangular.
 116. The array of claim 92, whereinthe detector layer is part of a CMOS sensor.
 117. The array of claim 92,further comprising an analyte disposed within the nanoscale emissionvolume.
 118. The array of claim 117, wherein the analyte comprises abiological sample.
 119. The array of claim 118, wherein the biologicalsample comprises a nucleic acid.
 120. The array of claim 118, whereinthe biological sample comprises a polymerase enzyme.
 121. The array ofclaim 92, wherein the array comprises at least 1,000, at least 10,000,at least 100,000, at least 1,000,000, or at least 10,000,000 nanoscaleemission volumes.